A direct test is performed to confirm the presence of cells with amastigotes in a dermis scrape[46,47]; the sample must be, in so far as it is possible, free of blood, cellular detritus or pus. If the patients present several lesions, the sample must be taken from the one with the shortest time of evolution, the biggest induration area and/or the least purulent discharge. If the lesion has a scab it must be removed to improve the quality of the sample. The sample can be taken from the active edge or from the bottom of the ulcer. When the sample is taken from the active edge asepsis must be realized with alcohol at 70% and hemostasis then performed using the first and second fingers to make sure that there is no blood in the sample. A small incision of about 3mm in length and depth is made with a scalpel in the active edge parallel to the edge of the ulcer. With the sharp side of the scalpel, a scrape is done in the dermic wall of the incision to obtain tissue. The extracted material is extended on two microscope slides. The sample is dried at room temperature, and then fixed with methanol and stained with Giemsa, Wright or Field. Using immersion oil the sample is observed under a microscope with a 100X lens, the presence of amastigotes of Leishmania assessed and their structure verified (nucleus, kinetoplast and cell membrane). If the sample is taken from the center of the lesion the same hemostatic technique must be used, the scab removed, and the bottom of the ulcer well cleaned using the sharp side of the scalpel to prevent the presence of cellular detritus and/or purulent material. This procedure must be continued until the granulomas are seen at the bottom. With the same technique used to process the samples from the active edge the presence of amastigotes of Leishmania is verified.